Cytochrome P450 monooxygenases (P450s) form a superfamily of haemoproteins which catalyse the metabolism of a wide range of compounds. They catalyse the oxidation of lipophilic chemicals through the insertion of one atom of molecular oxygen into the substrate (Porter & Coon (1991) J. Biol. Chem. 261, 13469-13472). Mammalian P450s catalyse the metabolism of endogenous and exogenous compounds, including steroids, therapeutic drugs and carcinogens (Guengerich (1987) in “Enzymology of rat liver cytochromes P450”, ed. Guengerich, F. P., CRC Press, Boca Raton Fla., Vol. 1, pp 1-54; Guengerich & Shimada (1991) Chem. Res. Toxicol. 4, 391-407; Gonzalez (1992) Trends in Pharmacol. Sci. 12, 346-352). The different mammalian P450s exhibit a unique but overlapping substrate specificity and display a high regio- and stereoselectivity (Crespi et al (1993) Toxicology 82, 89-104). Based on their major functions, mammalian P450s can be subdivided into two major classes: those involved primarily in the metabolism of steroids and bile acids, and those which mainly metabolise xenobiotics. The xenobiotic-metabolising P450s are typically located in the endoplasmic reticulum of certain mammalian cells such as liver cells and are termed microsomal P450s.
The compounds metabolised by the latter group of P450s include therapeutic drugs such as cyclosporin, nifedipine and debrisoquine as well as carcinogens such as polycyclic aromatic hydrocarbons, nitrosamines and arylamines. To be catalytically active, microsomal P450s require a supply of electrons which are shuttled from NADPH via the FMN and FAD prosthetic groups of NADPH-cytochrome P450 oxidoreductase (P450 reductase; EC 1.6.2.4) (Smith et al (1994) Proc. Natl. Acad. Sci. USA 91, 8710-8714).
Comparison of the primary structures of P450s indicates that they are structurally related to each other and are most likely derived from a common ancestor. Based on their primary structure the P450s are classified into families such as CYP1, CYP2 etc (Nelson et al (1996) Pharmacogenetics 6, 1-42).
Because of the importance of mammalian P450s in the metabolism of therapeutic compounds and carcinogens, attempts have been made to express mammalian P450s in heterologous systems. For example, mammalian cells have been used to express P450s heterologously as described by Doehmer et al (1988) Proc. Natl. Acad. Sci. USA 85, 5769-5773; Aoyama et al (1990) Proc. Natl. Acad. Sci. USA 87, 4790-4793; and Crespi et al (1991) Carcinogenesis 12, 355-359.
Yeast cells have also been used for the heterologous expression of cytochrome P450, for example by Renaud et al (1993) Toxicology 82, 39-52 and Bligh et al (1992) Gene 110, 33-39.
More recently mammalian P450s have been expressed in Escherichia coli. 
Gillam et al (1993) Arch. Biochem. Biophys. 305, 123-131 describes the expression of modified human cytochrome P450 3A4 in E. coli and purification and reconstitution of the enzyme.
Barnes et al (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601 describes the expression and enzymatic activity of recombinant cytochrome P450 17α-hydroxylase in E. coli. 
Larson et al (1991) J. Biol. Chem. 266, 7321-7324 describes that expression of cytochrome P450 IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity.
Shimada et al (1994) Carcinogenesis 15, 2523-2529 describes the activation of procarcinogens by human cytochrome P450 enzymes expressed in E. coli. 
Shet et al (1993) Proc. Natl. Acad. Sci. USA 90, 11748-11752 describes the enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase.
Shet et al (1995) Arch. Biochem. Biophys. 318, 314-321 describes some properties of a recombinant fusion protein containing the haem domain of human P450 3A4 and the flavin domains of rat cytochrome P450 reductase.
Jenkins & Waterman (1994) J. Biol. Chem. 269, 27401-27408 describes that flavodoxin and NADPH-flavodoxin reductase from E. coli support bovine cytochrome P450 c17 hydroxylase activities.
Fisher et al (1992) FASEB J. 6, 759-764 describes the expression of human cytochrome P450 1A2 in E. coli. 
Fisher et al (1992) Proc. Natl. Acad. Sci. USA 89, 10817-10821 describes the expression in E. coli of fusion proteins containing the domains of mammalian cytochromes P450 and NADPH-P450 reductase flavoprotein.
Chun & Chiang (1991) J. Biol. Chem. 266, 19186-19191 describes the expression of cholesterol 7α-hydroxylase cytochrome P450 in E. coli. 
Richardson et al (1995) Arch. Biochem. Biophys. 323, 87-96 describes the expression of human and rabbit cytochrome P450s of the 2C subfamily in E. coli. 
Gillam et al (1995) Arch. Biochem. Biophys. 319, 540-550 describes the expression of cytochrome P450 2D6 in E. coli. 
Dong & Porter (1996) Arch. Biochem. Biophys. 327, 254-259 describes a study in which P450 reductase containing an N-terminal fusion to an ompA signal peptide is co-expressed in E. coli with human P450 2E1 in which the second codon of the P450 (serine) is replaced with an alanine; no other changes to the P450 were made. In vivo activity with whole cells could not be demonstrated.
WO 94/01568 describes the expression of P45017α-hydroxylase in E. coli and also its fusion to a P450 reductase enzyme domain and expression of the fusion protein in E. coli. P450 enzyme hybrids, incorporating the N-terminal nine amino acids from bovine P45017α-hydroxylase are also disclosed.
U.S. Pat. No. 5,240,831 describes expression of P45017α-hydroxylase in E. coli in a biologically active form without the need for co-expression or admixture of a cytochrome P450 reductase.
Gillam et al (1995) Arch. Biochem. Biophys. 317, 374-384 describes the expression of cytochrome P450 3A5 in E. coli. 
Gillam et al (1994) Arch. Biochem. Biophys. 312, 59-66, describes the expression of modified human cytochrome P450 2E1 in E. coli. 
Shet et al (1994) Arch. Biochem. Biophys. 311, 402-417 describes a recombinant fusion protein expressed in E. coli containing the domains of bovine P450 17A and rat NADPH-P450 reductase.
Josephy et al (1995) Cancer Res. 55, 799-802 describes the bioactivation of aromatic amines by recombinant human cytochrome P450 1A2 expressed in Salmonella typhimurium. 
Despite the extensive efforts to express an effective eukaryotic, particularly mammalian, P450 monooxygenase enzyme system in bacteria to date no system which is capable of metabolising compounds in whole cells has been devised. This is particularly the case for xenobiotic-metabolising P450s which require a P450 reductase for enzymatic activity. More particularly, no bacterial cell system has previously been devised which allows cytochrome P450 and cytochrome P450 reductase to form a functional cytochrome P450 monooxygenase system when expressed separately in the same bacterial cell. Similarly, despite considerable efforts to produce bacteria which express a high level of a eukaryotic xenobiotic-metabolising P450, no satisfactory system has so far been devised.
One object of the invention is to provide superior systems for expressing P450s in a functional form in intact bacterial cells.
A further object of the invention is to provide an improved system for expressing P450s whether or not with P450 reductase.
Bacterial systems which express a functional P450 enzyme system in whole cells are useful as “bioreactors” or they may find uses in drug-testing or carcinogen-testing systems or as biosensors or in environmental remediation or in the production of hormones and so on. The expression of eukaryotic P450s at high levels in bacteria provides a source of P450 for structural studies.